DNA analysis of low- and high-density fractions defines heterogeneous subpopulations of small extracellular vesicles based on their DNA cargo and topology.

Extracellular vesicles have the capacity to transfer lipids, proteins, and nucleic acids between cells, thereby influencing the recipient cell's phenotype. While the role of RNAs in EVs has been extensively studied, the function of DNA remains elusive. Here, we distinguished novel heterogeneous subpopulations of small extracellular vesicles (sEVs) based on their DNA content and topology. Low- and high-density sEV subsets from a human mast cell line (HMC-1) and an erythroleukemic cell line (TF-1) were separated using high-resolution iodixanol density gradients to discriminate the nature of the DNA cargo of the sEVs. Paired comparisons of the sEV-associated DNA and RNA molecules showed that RNA was more abundant than DNA and that most of the DNA was present in the high-density fractions, demonstrating that sEV subpopulations have different DNA content. DNA was predominately localised on the outside or surface of sEVs, with only a small portion being protected from enzymatic degradation. Whole-genome sequencing identified DNA fragments spanning all chromosomes and mitochondrial DNA when sEVs were analysed in bulk. Our work contributes to the understanding of how DNA is associated with sEVs and thus provides direction for distinguishing subtypes of EVs based on their DNA cargo and topology.

Association of polymorphisms in genes coding for antioxidant enzymes and human male infertility.

PURPOSE: Although oxidative stress is thought to be an important cause of male infertility, primarily due to DNA and cell membrane damage, little is known about the genetic causes underlying suboptimal function of the seminal enzymatic antioxidant system. The aim of this study was to investigate the relationship of four potentially functional polymorphisms associated with oxidative stress pathway genes (superoxide dismutase-SOD2 lle58Thr and SOD2 rs4880, catalase-CAT C-262T, glutathione peroxidase 1-GPX1 Pro200Leu) and two null variants of the glutathione S transferase (GSTT and GSTM) genes and infertility risk. METHODS: A case control study was conducted on 313 infertile patients and 80 fertile donors. Each ejaculate was subjected to a seminal analysis that included the classical parameters seminal volume, sperm concentration, sperm motility, and sperm morphology, as well as sperm DNA fragmentation (patients only). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR multiplex methods were carried out for genotyping. RESULTS: Statistically significant differences were found between fertile donors and infertile patients for SNP CAT C-262T; the CC genotype was related with a twofold increased risk of infertility (odds ratio [OR] = 2.262; 95% confidence interval [CI] = 1.369-3.733; P = 0.001), whereas the CT genotype was associated with a protective effect (OR = 0.401; 95% CI = 0.241-0.667; P = 0.001). Surprisingly, the SOD2 Ile58ssThr SNP was not represented in the sample population, so its frequency in the current population frequenting fertility clinics in Madrid may be very low. CONCLUSIONS: Our results suggest that the CAT SNP C-262T is potentially associated with an increased risk of male infertility.

DNA Damage and Repair in Human Reproductive Cells.

The fundamental underlying paradigm of sexual reproduction is the production of male and female gametes of sufficient genetic difference and quality that, following syngamy, they result in embryos with genomic potential to allow for future adaptive change and the ability to respond to selective pressure. The fusion of dissimilar gametes resulting in the formation of a normal and viable embryo is known as anisogamy, and is concomitant with precise structural, physiological, and molecular control of gamete function for species survival. However, along the reproductive life cycle of all organisms, both male and female gametes can be exposed to an array of "stressors" that may adversely affect the composition and biological integrity of their proteins, lipids and nucleic acids, that may consequently compromise their capacity to produce normal embryos. The aim of this review is to highlight gamete genome organization, differences in the chronology of gamete production between the male and female, the inherent DNA protective mechanisms in these reproductive cells, the aetiology of DNA damage in germ cells, and the remarkable DNA repair mechanisms, pre- and post-syngamy, that function to maintain genome integrity.

Impact of polymorphism in DNA repair genes OGG1 and XRCC1 on seminal parameters and human male infertility.

The DNA repair capacity in the mature spermatozoa is highly compromised due to the base-excision repair (BER) route being truncated. In the mature spermatozoa, only the first enzyme of the route (OGG1) is present. Consequently, reduced activity of the enzymes of the BER route both during spermatogenesis and in the mature spermatozoa may be detrimental for fertility. The objective of our study was to investigate the correlation between two representative SNPs of those enzymes, SNPs OGG1 Ser326Cys (rs1052133) and XRCC1 Arg399Gln (rs25487) and male infertility. A total of 313 seminal samples from infertile patients and 80 from donors with proven fertility were included in the study. All samples were subjected to a regular sperm analysis and genotyped using the PCR-RFLP system. We found significant differences in the genotype frequencies between patients and donors for the XRCC1 Arg399Gln polymorphism (χ2(2) = 8.7, p = 0.013), with the Gln allele showing a protective role and for the OGG1 Ser326Cys polymorphism between normozoospermic and non-normozoospermic patients (χ2(2) = 12.67, p = 0.002) with the Cys allele showing a detrimental effect over concentration. In conclusion, our study shows that polymorphisms in the genes coding for the DNA damage repair enzymes may be associated with poor sperm parameters and male infertility.